1. Field of the Invention
This invention, in part, relates to the coupling of certain anti-craving compositions and specific genotyping of a number of genes all involved in neurotransmitter function of reward behavior. An aspect of this invention is the understanding of the involvement of how certain established neurotransmitters work in concert to activate neuropathways in the meso-limbic system of the brain leading to feelings of well being, and the development of compositions that affect these neuropathways. This invention, in part, relates to the utilization of precursor amino acids and certain herbal compounds to enhance attentional processing and memory as well increase focus in healthy individuals, as well as to enhance weight loss and control overeating. Disclosed are various diagnostic methods of neurological disorders and behaviors utilizing genetic polymorphisms of neurotransmitter genes, and therapeutic methods of treatment of patients so identified using the compositions of the invention. Also disclosed are diagnostic methods for polygenic traits.
2. Description of Related Art
During the past several decades, research on the biological basis of chemical dependency has been able to establish some of the brain regions and neurotransmitters involved in reward. In particular, it appears that the dependence on alcohol, opiates and cocaine relies on a common set of biochemical mechanisms (Cloninger, 1983; Blum, 1978; Blum, 1989). A neuronal circuit deep in the brain involving the limbic system and two regions called the nucleus accumbens and the globus pallidus appears to be critical in the expression of reward of people taking drugs (Wise and Bozarth, 1984). It has been demonstrated that the chronic use of cocaine, morphine and alcohol results in several biochemical adaptations in the limbic dopamine system (Ortiz et al., 1996).
The mesolimbic dopamine system connects structures high in the brain, especially the orbiofrontal cortex (in the prefrontal area behind the forehead) with the amygdala in the brain's center, and with the nucleus accumbens, which has been proven in animal research to be a major site of activity in addiction. The various brain pathways involved in multiple addictions converge on certain dopaminergic receptors (D1, D2, D3, D4, D5) where the D2 site seems to be most prominent. Although each substance of abuse appears to act on different parts of the circuit, the end result is the same: dopamine is released in the nucleus accumbens and the hippocampus (Koob and Bloom, 1988). Dopamine appears to be the primary neurotransmitter of reward at these reinforcement sites.
Abnormalities in dopamine metabolism have been implicated in several behaviors, i.e. sexual disorders (Gessa and Tagiamonte, 1975), mania (Goodwin and Jamison, 1990), schizoid behaviors (Carlsson, 1978; Snyder, 1976), ADHD (Shaywitz et al., 1976), conduct disorder or aggression (Rogeness et al., 1986; Valzelli, 1981; King, 1986), alcohol abuse (Blum et al., 1990) and stuttering. In addition, haloperidol, a DRD2 receptor antagonist, has been reported to be effective in the treatment of some stutters (Murray et al., 1977; Prins et al., 1980). While serotonergic mechanisms have been most often implicated for obsessive- compulsive behaviors, abnormalities in dopamine have also been considered (Austin et al., 1991; Delgado et al., 1990). Abnormal circuits involving the thalamus, basal ganglion and frontal lobes have been implicated in obsessive-compulsive disorder (Baxter et al., 1992; Rauch et al., 1994; Modell et al., 1989) and dopamine is a major neurotransmitter especially in the striatum and frontal lobes.
Defects in central noradrenergic mechanisms have been frequently implicated in the etiology of attention-deficit hyperactivity disorder. A significant increase in plasma noradreneline (NA) in ADHD children with reading and other cognitive disabilities compared to ADHD children without cognitive disabilities has been demonstrated (Halperin et al., YEAR). They proposed that the ADHD+cognitive disabilities was associated with NA dysregulation affecting the parietal/temporal lobe attention centers. Since these brain areas are in proximity to auditory and linguistic processing regions, this could account for the comorbid cognitive disabilities. From a clinical perspective, the significant improvement in symptoms that often occurs following treatment with clonidine (Hunt et al., 1985; Comings et al., 1990) implies a role of NA in at least some ADHD. Clonidine is a presynaptic a.sub.2 -noradrenergic receptor agonist that results in the inhibition of release of noradrenaline into the synapse (Starke et al., 1974).
It has been proposed that NA and the locus coeruleus (LC) play a role in arousal and vigilance, critical aspects of attention (Aston-Jones et al., 1984). It has been proposed that stress tolerance and good performance on tasks were related to low basal or tonic levels of catecholamines and to higher acute releases during mental stress (Dienstbier, 1989). The opposite may occur in ADHD, with an increased baseline tonic stimulation of NA and a decreased release of catecholamines during stress (Pliszka et al., 1996). To test the hypothesis that NA defects are involved in ADHD, a number of studies of CSF, plasma and urinary excretion of the NA metabolite (3-methoxy-4-hydroxyphenylglycol (MHPG) have been performed. Some show that ADHD patients have lower rates of MHPG excretion than controls (Oades, 1987; Shekim et al., 1983; Shekim, Dekirmenjian, and Chapel, 1997; Yu-cum and Yu-feng, 1984) while others show no change (Rapoport et al., 1978; Zametkin et al., 1985) or an increase in NA turnover (Khan and Dekirmenjian, 1981). Epinephrine levels have been reported to be significantly lower (Hanna et al., 1996; Klinteberg and Magnusson, 1989; Pliszka et al., 1994), or to show a blunted response to glucose ingestion (Girardi et al., 1995) in ADHD subjects compared to controls. Norepinephrine is converted to epinephrine (adrenaline) by phenylethanolamine N-methyl-transferase coded by the PNMT gene.
A model of ADHD based on failure of epinephrine to tonically inhibit NA neurons in the locus coeruleus. d-amphetamine and desipramine, both of which are commonly used in the treatment of ADHD, lead to a significant decrease in the excretion of MHPG has been proposed (Mefford and Potter, 1989; Shekim et al., 1979). However, methylphenidate (Ritalin) the most commonly prescribed medication for the treatment of ADHD does not result in a decrease in MHPG excretion (Zametkin et al., 1985) and other medications that reduce MHPG excretion, such as fenfluramine (Donnelly et al., 1989), are not effective in the treatment of ADHD. These observations are consistent with the presence of several types of ADHD and an involvement with multiple neurotransmitters and genes.
It has been proposed that NA and adrenergic .alpha.2-receptors played a role in some forms of ADHD through a dysregulation at the LC of the posterior cortical attention system (Posner and Peterson, 1990; Pliszka 1996) of the parietal/temporal lobes, and that a second form of ADHD was due to dopaminergic defects that primarily affected the prefrontal lobe attentional system which was associated with impulsivity and disorders of executive dysfunction. Several dopaminergic genes, such as the dopamine D.sub.2 receptor (DRD2) (Comings et al., 1991), dopamine D.sub.4 receptor (DRD4) (Lahoste et al., 1996); and dopamine transporter (DAT1) (Cook et al., 1995; Comings et al.,. 1996; Gill et al., 1997; Waldmaqn et al., 1996) genes have been found to be associated with ADHD.
It has been reported that boys with ADHD and reading disabilities had significantly higher plasma MHPG levels than boys with ADHD only (Haperin et al, 1993; Halperin et al., 1997). In the latter study, they also demonstrated a significant negative correlation between plasma MHPG levels and the WISC-R verbal IQ, and the reading, spelling and arithmetic problems assessed by the WRAT-R (Wide-Range Achievement Test-Revised). This distinction was consistent with prior studies of others suggesting that ADHD with cognitive disabilities was a distinct subtype of ADHD (August and Garfinkel, 1989; McGee et al., 1989; Pennington et al., 1993). It has also been suggested that the type of attention deficit associated with parietal lobe defects tends to be a selective attention deficit (Posner and Peterson, 1990; Dykman et al., 1979; Richards et al., 1990).
It has been proposed that ADHD+cognitive disorders was due to a dysregulation of NA metabolism of the LC involving adrenergic .alpha.2 receptors, and primarily affected the posterior attention system of the parietal cortex (Halperin et al., YEAR) Since these brain areas are in proximity to auditory and linguistic processing regions, this could account for the comorbid cognitive disabilities. It would be a mistake to assume that these are pure forms since ADHD is a polygenic disorder (Comings et al., 1996), and most individuals are likely to have inherited genes for both types. Studies in primates show that NA and defects in adrenergic .alpha.2 receptors also play a role in prefrontal lobe cognitive defects (Arnsten, 1997).
Various studies have indicated the involvement of the dopamine receptor in addictive behaviors. Cocaine patients show a drop in those neuronal activity levels that is consistent with a lessened ability to receive dopamine (Volkow et al., 1993). Neurons with D2 dopamine receptors were shown to become 25% smaller, and lost much of their ability to receive small amounts of dopamine from nearby neurons in morphine addicted rats (Nestler et al, 1996). Decreases in D2 receptors observed in opiate-dependent subjects have been suggested to indicate that the subjects had low D2 receptors prior to when they began abusing drugs, and that this reduction may have made them more vulnerable to drug self-administration (Wang et al., 1997).
Although the system of neurotransmitters involved in the biology of reward is complex, at least three other neurotransmitters are known to be involved at several sites in the brain: serotonin in the hypothalamus, the enkephalins (opioid peptides) in the ventral tegmental area and the nucleus accumbens, and the inhibitory neurotransmitter GABA in the Substantia nigra, ventral tegmental area and the nucleus accumbens (Stein and Belluzzi, 1986; Blum and Kozlowski, 1990). Interestingly, the glucose receptor is an important link between the serotonergic system and the opioid peptides in the hypothalamus. An alternative reward pathway involves the release of norepinephrine in the hippocampus from neuronal fibers that originate in the locus coeruleus.
There is evidence that the opoidergic and dopaminergic systems are anatomically and functionally interconnected, suggesting a role for the endogenous opioidergic system in mediating the effects of ethanol and other drugs on brain dopaminergic pathways associated with reward. Dopamine antagonists and lesions of the dopaminergic pathways in the brain affect pre-proenkephalin A activity (Morris et al., 1988; Normand et al., 1988). Behavioral, pharmacological and neurochemical studies implicate the opioidergic and dopaminergic systems in the reinforcing effects of ethanol and other drugs of abuse (Blum et al., 1976a, b; Blum et al., 1982a; Blum et al., 1977; Blum et al., 1973; Koob and Bloom, 1988; Weiss et al., 1993). Animal studies show that opiate receptor agonists increase preference for ethanol, whereas antagonists of these receptors reduce ethanol consumption (Blum et al., 1975; Le et al., 1993). Further, studies on animals and human alcoholics suggest the effectiveness of the opiate receptor antagonist in reducing the positive reinforcing effects of alcohol consumption (O'Malley, 1992; Swift et al., 1994; Blum et al., 1975; Volpicelli et al., 1992). Moreover, ethanol-induced increase of brain dopamine levels in animals is blocked by both opiate receptor antagonists naloxone and naltrexone (Widdowson and Holman, 1992; Benjamin et al., 1993). A recent review by Gianoukalis and de Waele (1994) support the role of endogenous opioids and drugs of abuse (i.e. alcohol).
In a normal person, these neurotransmitters work together in a cascade of excitation or inhibition between complex stimuli and complex responses, leading to a feeling of well being, the ultimate reward. In the cascade theory of reward, a disruption of these intercellular interactions results in negative emotions. Genetic anomalies, including certain polymorphisms, prolonged stress or longer term abuse of psychoactive drugs (including glucose) can lead to a self-sustaining pattern of abnormal cravings in both animals and human beings (Blum, 1991).
Pharmacological actions (bromocryptine, bupropion and N-propylnorapomorphine) are partly determined by the individual's dopamine D2 genotype. A1 carriers of the DRD2 gene are pharmacologically more responsive to D2 agonists. One study has already shown that the direct microinjection of the D2 agonist N-propylnorapomorphine into the rat nucleus accumbens significantly suppresses the animal's symptoms after withdrawal from opiates, while dopamine per se suppresses alcohol withdrawal symptoms (Harris and Aston-Jones, 1994; Blum et al., 1976b). In this regard, there is evidence for dopamine/endogenous opioid peptide interactions in the nucleus accumbens and elsewhere in the brain, and it may be that overstimulation of the opioid peptide system by exogenous opiates leads to decreases in dopamine function (Pothos et al., 1991). When compared to normal non-alcohol preferring rats, alcohol preferring rats have fewer serotonin neurons in the hypothalamus, higher levels of enkephalins in the hypothalamus (because less is released), more GABA neurons in the nucleus accumbens and a lower density of D2 receptors in certain areas of the limbic system (McBride et al., 1995; Smith et al., 1997; and McBride et al., 1997).
Clinical trials have demonstrated that when amino-acid precursors of certain neurotransmitters (serotonin and dopamine) and D-phenylalanine, a substance that promotes enkephalin activity by inhibiting enzymatic cleavage (U.S. Pat. Nos. 4,761,429 and 5,189,064) are administered to probands with either SUD or carbohydrate bingeing, was found to reduce craving, reduce incidence of stress, reduce relapse rates, and also increase the likelihood of recovery.
A number of laboratories have pursued the association between certain genes and various behavioral disorders, including linkage of the dopamine D2 receptor alleles with a number of impulsive-compulsive-addictive behaviors. Little is known about the resultant expression of polymorphisms linked to either the DAT.sub.1 10/10 allele and the D.beta.H B.sub.1 allele except studies showing increased dopamine transporter sites in Tourette's Disorder patients by SPECT scanning techniques (Malison et al., 1995, Tiihonen et al., 1995). ADHD, Tourette syndrome, conduct disorder, ODD, dyslexia, learning disorders, stuttering, drug dependence and alcoholism all show a male predominance. The molecular genetic studies of the DRD2, D.beta.H, DAT (Comings et al., 1996a) and clinical genetic studies (Comings, 1994b; 1994c; 1995b; Bierderman et al., 1991; Comings and Comings, 1987), indicate these are etiologically related spectrum disorders. Defects in neurotransmitters has been advocated as involved in alcoholism (Blum, 1991). Studies of genes involved in neurological pathways are described below.
Androgent Receptor gene Specific mutations of the AR gene have been reported to cause a wide range of types of androgen insensitivity syndromes (Gottlieb et al., 1977). In addition, two sets of polymorphic tricnucleotide repeat sequences, CAG (Edwards et al., 1992) and GGC (Sleddens et al., 1993; Sleddens et al., 1992), resulting in polyamino acid tracts in the protein, are present in the first exon of the AR gene. When highly expanded, from 43 to 65 times, the CAG trinucleotide repeat has been shown to cause X-linked spinal muscular atrophy (La Spada et al., 1991). The repeat length in the normal population is 11 to 31 times (Edwards et al., 1992).
The non-highly expanded alleles of micro- and minisatellites present in the normal population, might play a direct role in the regulation of genes. This was based on the observation that most short tandem repeats are associated with the formation of Z-DNA (Schroth et al., 1992), and Z-DNA has repeatedly been implicated in various aspects of gene regulation (Rich et al., 1984; Hamada et al., 1982; Wolff et al., 1996). Since the amount of Z-DNA formed is highly sensitive to the length of the repeats (Schroth et al., 1992), it was suggested that the size of the repeat alleles could themselves be related to phenotypic effects (Comings, 1997).
Some (Olweus et al., 1988; Mattsson et al., 1980; Schiavi et al., 1984; Kreuz and Rose, 1972) but not all (Bradford and McClean, 1984; Schaal et al., 1998) studies suggest a correlation between aggressive behavior and plasma testosterone levels. Aggressive conduct disorder is often a comorbid condition in subjects with TS and ADHD (Comings, 1995; Stewart et al., 1981; Biederman and Sprich, 1991) and there is a high degree of comorbidity between TS and ADHD (Comings and Comings, 1984, 1990; Knell and Comings, 1993).
Dopamine D.sub.1 Receptor Gene (DRD1) Sequencing of the DRD1 gene in controls and in patients with schizophrenia, manic-depressive disorder and alcoholism, has failed to identify exon mutations that produce an effect on the phenotype and linkage studies in schizophrenia and TD (Jensen, 1993k; Gelertner et al., 1993k). The D.sub.1 receptors in frontal cortex may play a role in memory (Comings et al., 1997k; Williams et al., 1995k). The opposing effect of the D.sub.1 and D.sub.2 receptor agonists on cocaine seeking behavior in rats have been reported (Self et al., 1996). TD probands, smokers, and pathological gamblers, were consistent with negative heterosis, in that the most consistent difference was a relative decrease in the frequency of 12 heterozygotes and an increase in 11 and 22 homozygotes of the Dde 1 polymorphism (Comings et al. (1996)). By contrast, positive heterosis was present at the DRD2 gene, with quantitative scores being highest for 12 heterozygotes and lowest for 11 and 22 homozygotes. While the results for ADHD at the DRD1 locus alone was not significant, there was a significant additive effect of examining the presence of negative heterosis at the DRD1 gene and/or positive heterosis at the DRD2 gene (Comings et al., 1997k).
Dopamine D2 Receptor Gene (DRD2) Previous studies have shown a significantly increased prevalence of the D2A1 allele in individuals with ADHD, TS ,CD and SUD (Comings et al., 1991). Since each of these disorders is characterized by a poor response to stress and many criteria for the diagnosis of PTSD have many symptoms in common with ADHD and The National Vietnam Veterans Readjustment Study (Kulka et al., 1990) reported a significant correlation between PTSD and a history of childhood symptoms consistent with ADHD and CD. Subjects on an addiction treatment unit who had been exposed to severe combat conditions in Vietnam were screened for posttraumatic disorder (PTSD), and showed an correlation of individuals with PTSD as carrying the D2A1 allele (Comings, et al., 1996k).
An association between ADHD and the TaqA1 allele of the DRD2 gene has been detected (Comings et al., 1991k). The stimulant methlphenidate increased regional blood flow while in others it decreased blood flow. The changes in frontal, temporal and cerebellar metabolism were related to the density of D2 receptors--the higher the density the greater the increases in blood flow. Methylphenidate decreased the relative metabolic activity in the basal ganglia. These results are consistent with the idea that genetic defects in dopamine metabolism, resulting in a dopaminergic state in the limbic system and frontal lobes result in a compensatory increase in dopaminergic activity in the basal ganglia, and that methylphenidate reverses these through a combination of enhancing brain dopamine activity in dopaminergic through its inhibition of the dopamine transporter (Volkow et al., 1996k), with a secondary decrease in dopaminergic activity in the basal ganglia and decrease in basal ganglia blood flow. These studies are also consistent with the results of others (Castellanos et al., 1996k). showing a positive correlation between the response to methylphenidate and CSF levels of HVA, a metabolite of dopamine whose levels in the CSF are related to D2 receptor density (Jonsson et al., 1996k). Methylphenedate consistently increased cerebellar metabolism, despite the paucity of D2 receptors in this structure (Volkow et al., 1996k; Hall et al., 1994k). This is consistent with the increasing evidence that the cerebellum play an important role in attention, learning and memory (Leiner et al., 1989k). An association between the A1 genotype and regional blood flow has been reported. The TaqI D2 A1 carriers showed a significantly lower relative glucose metabolism in the putamen, nucleus accumbens, frontal and temporal gyri and medial prefrontal, occipito-temporal and orbital cortices than those with the A22 genotype (Nobel et al., 1997). The TaqI D2 A1 carriers had a significantly decreased dopamine D2 receptor B.sub.max in the basal ganglia (Noble et al., 1991k). Enkephalin increases blood flow in similar regions as methylphenidate and may therefore involve a dopaminergic mechanism (Blum et al., 1985k). A significant decrease in dopamine D2 receptor density was measured in individuals with detachment, social isolation, and lack of intimate friendships (Farde et al., 1997k).
Though the DRD2 gene polymorphisms have been associated with a number of psychological disorders, but no association was found between certain psychopathy in incarcerated drug users (Smith et al., 1993). The reports of an association between alcoholism and the DRD2 allele have been quite variable, an association between the D.sub.2 A1 allele and polysubstance/drug abuse has been found (Smith, et al., 1992, Noble, et al., 1993, O'Hara, et al., 1993, Comings, et al., 1994, U.S. Pat. No. 5,210,016, U.S. Pat. No. 5,500,343). After the first association of the DRD2 A1 and severe alcoholism (Blum et al., 1990), several groups were unable to replicate the observation. Two possible reasons were suggested: first, inadequate screening of controls for alcohol, drug and tobacco abuse; and second, sampling errors in terms of characterization of alcoholics for chronicity and severity of disease (Blum et al., 1997; Bolos et al., 1990; Gelertner et al., 1991; Schwab et al., 1991; Turner et al., 1992; Cook et al., 1992; Goldman et al., 1992; Goldman et al, 1993; Suarez et al., 1994).
Dopamine plays a role as a modulator of many different behaviors (LeMoal and Simon, 1991), and numerous studies have reported a significant association between alleles of the DRD2 gene and cocaine addiction, polysubstance abuse, smoking, attention deficit hyperactivity disorder (ADHD), Tourette syndrome, visual-perceptual disorders, conduct disorder, posttraumatic stress disorder, pathological gambling, and compulsive eating (Blum et al., 1995; Blum et al., 1996). Despite these associations, sequencing studies have failed to find any mutations in the exons that could explain these findings. These findings could be explained if the D.sub.2 A1 allele was in linkage disequilibrium with an unknown non-exon mutation that played a role in the regulation of DRD2 function (Comings et al., 1991). Additionally the severity of alcoholism and the type of controls used have been reported as important determinants of DRD2A1 allele association with alcoholism (Noble et al., 1994; Geijer et al., 1994; Parsian et al, 1991; Blum et al., 1992; Blum et al., 1990; Lawford et al., 1997).
Sib pair linkage analyses conducted in families multiply affected by alcoholism, using both the TaqI "A" RFLP and a microsatellite repeat polymorphism at the DRD2 locus, indicated a significant correlation with this locus and the liability to develop heavy drinking. A corresponding mutation in the DRD2 gene has not been found, the effect may arise from a closely linked locus-outside the DRD2 gene itself (Cook et al., 1996). A single point mutations in exon 8 of the DRD2 gene in alcohol-dependent patients has been demonstrated (Finch et al., 1995), while others report no structural mutations in the coding regions of the DRD2 gene (Gejman et al., 1993).
The DRD.sub.2 gene A.sub.1 allele has been found to associate with a number of behaviors including severe alcoholism, polysubstance dependence, crack/cocaine addiction, tobacco smoking, pathological gambling, lack of a major depressive episode, and carbohydrate bingeing or generalized to DSM- IV substance use disorder (Blum et al., 1996e; Blum et al., 1995b; Comings et al., 1996c). The MCMI-II assessed schizoid/avoidant cluster compared to other Axis II diagnostic clusters (antisocial, narcissistic, paranoid) significantly correlated with alcohol abuse scales (Corbisiero et al., 1991). Clusters of patients with MCMI-II elevations that indicated schizoid and avoidant qualities tended to stay in treatment fewer days and relapsed earlier (Fals-Stewart, 1992). High scores of schizoid/avoidant cluster correlated with inpatient male alcoholics (Matano et al., 1994) and cocaine dependent patients (Kranzler and Satel, 1994). Schizoid/avoidant behaviors including low levels of sensation were found to consume more alcohol and to have higher MAST scores than patients with high levels of sensation (Ohannessian and Hesselbrock, 1995); and avoidant personality is significantly correlated in subjects with severe binge eating disorder (Yanovski et al., 1993).
Molecular heterosis at the dopamine receptor genes was indicated in healthy individuals and alcoholics. Cerebrospinal fluid levels of monoamine metabolites consisting of HVA for dopamine, 5-HIAA for serotonin, and MHPG for norepinephrine levels were compared in healthy volunteers to the DRD2 TaqI A1A2 and B1B2 genotypes. The results indicate a statistically significant difference in the means of the 1,1+1,2 homozygotes vs. the 1,2, but not when analyzing the 1,1+1,2 vs. the 2,2 genotypes and for 1 vs. the 2 alleles. The TaqI B1B2 polymorphism gave virtually identical results (Jonsson et al., 1996). In contrast, CSF HVA levels and the DRD2 TaqIA1/A2 polymorphism were examined in Finnish and American alcoholics, and no association was found when examining the 1 vs. 2 alleles, and not the 1,1+1,2 vs. the 2,2 genotypes (Goldman et al., 1992).
Heterosis at the DRD2 gene was indicated by comparison of the CSF levels of HVA, to the DRD2 genotype using TaqI polymorphism (Jonsson et al., 1996k). In a profile for the inattention score of TD subjects, the 12 heterozygotes showed the highest inattention score subjects who were 12 heterozygotes had the lowest levels of CSF HVA (Jonsson et al., 1996). The highest levels of HVA were seen in the 11 homozygotes, with the 22 homozygotes being intermediate. Some studies, but not all, showed a significantly lower level of CSF HVA in children (Shaywitz et al., 1979k) with ADHD and TD (Cohen et al., 1979k). A significant correlation was found between electrophysiological abnormalities and the DRD2 A1 allele (Blum et al., 1994k). These abnormalities are seen in ADHD subjects as well as children of alcoholics (Comings et al., 1991k; Noble et al., 1994k).
A positive association of the TaqA1 of the DRD2 gene to Attention Deficit Disorder (ADHD) and Tourette's was reported (Comings et al., 1991; Comings et al., 1996a), while others found no association with ADHD probands (Sunohara et al., 1996). ADHD probands showed a significant association with the 48 bp variant of the D4 gene, but not the DRD2, DRD3 or the serotonin transporter genes. The 7-fold repeat allele of the DRD4 occurred significantly more frequently in that children with ADHD. There is evidence for an association of the 7 repeat allele of the D4 receptor gene and novelty seeking (characterized as impulsive, exploratory, fickle, excitable, quick tempered and extravagant) (Epstein et al., 1996; Benjamin et al., 1996). The DRD2 A1 allele in cocaine dependent probands was associated with the opposite: low novelty seeking, characterized by reflective, rigid, stoic, slow-tempered, avoidant, as well as having enhanced withdrawal depression (Compton et al., 1996). Molecular genetic studies have found an association of the D.sub.2 dopamine receptor (DRD2) A1 allele with alcoholism and drug abuse (Blum et al., 1990). Reduced central dopaminergic function has been suggested in subjects who carry the A1 allele (A1.sup.+) compared with those who do not (A1.sup.-) (Nobel et al 1997). The genes responsible for alcoholism are unknown, although the many studies to date indicate a significant role for the DRD2 gene in more severe cases (Noble, 1993; Blum et al., 1995).
The DRD2 gene has been associated with the compulsive behavior (Comings and Comings, 1987b) and addictive, impulsive behaviors, including compulsive eating, gambling and smoking. (Self et al., 1996; Ogilvie et al., 1996; Blum et al., 1995b; Blum et al., 1996e). These behaviors have previously been reported to be associated with the DRD2 gene (Comings et al., 1993a; Noble et al., 1994d; Blum et al., 1996a; Comings et al., 1996c; Noble et al., 1994c; Noble, 1993; Comings et al., 1996e) in subjects distinct from the TS group.
Dopamine D2 receptor availability was significantly lower in alcoholics than in nonalcoholics, and was not correlated with days since last alcohol use (Volkow et al., 1997). The ratio DRD2 receptor to transporter availability was significantly higher in nonalcoholics than in alcoholics. Alcoholics showed significant reductions in D2 receptors (postsynaptic marker) but not in DA transporter availability (presynaptic marker) when compared with nonalcoholics. Because D2 receptors in striatum are mainly localized in GABA cells, these results provide evidence of GABAergic involvement in the dopaminergic abnormalities seen in alcoholics.
Dopamine D.sub.3 Receptor Gene (DRD3) Knockout mice missing the DRD3 gene are considerably more active than their litter mates with normal DRD3 genes (Williams et al., 1995k). Negative heterosis has been reported for schizophrenia at the DRD3 locus (Crocq et al., 1992k) have observed a significant decrease in DRD3 MscI 12 heterozygosity in TD (Comings et al., 1993j) and pathological gambling (Comings et al., 1996).
Dopamine D.sub.4 Receptor Gene (DRD4) In the Dopamine D.sub.4 receptor gene (DRD4), a 48 bp and 16 amino-acid repeat polymorphism within the DNA coding for the third cytoplasmic loop responsible for binding to guanine-nucleotide proteins (Van Tol et al., 1992k; Lichter et al., 1993k) has been reported. This DNA region is repeated 2 to 11 times, with the most common alleles being the 2, 4, and 7 repeat. The 7 allele demonstrates a blunted response to dopamine in regards to intracellular adenyl cyclase inhibition (Asghari et al., 1995k). Two independent studies of normal subjects have shown an association between the presence of the 7 allele and novelty seeking, a trait associated with impulsivity (Benjamin et al., 1996k; Ebstein et al., 1996k). One study failed to find such an association (Malhotra et al., 1996). A study ADHD children compared to controls, reported that more ADHD children carried at least one 7 allele compared to controls (LaHoste et al., 1996k). An association between the 7 allele of the DRD4 gene in TD has been reported (Grice et al., 1996k). Other work in this area is not equivocal (Spielman et al., 1993k).
Dopamine Transporter Gene (DAT1) The DAT1 gene marker frequencies at the vesicular transporter locus showed substantial heterogeneity in different Caucasian-Americans originating from different European countries, but no association with substance abuse was evident (Uhl et al., 1993; Persico et al., 1993). Distributions of the DAT1 VNTR alleles do not distinguish any substance user or control sample for psychostimulant abusers (Persico et al., 1996), however an association was observed with Japanese alcoholics (Muramatsu and Higuchi, 1995). The DAT1 gene has also been implicated as having a role in compulsive and addictive disorders. Since one of the modes of action of cocaine is to inhibit dopamine transporter function (Ritz, et al., 1992; Ritz, et al., 1990), it has been implicated in the biology of drug addiction, as well as other disorders including Parkinson's disease (Uhl, 1990) and Tourette's syndrome (Singer, et al., 1991).
Increased dopamine transporter sites in Tourette's syndrome was demonstrated using SPECT scanning techniques (Malison et al., 1995), and dopamine transporter receptor sites were significantly increased in violent alcoholics compared to non-violent alcoholics (Tiihonen et al., 1995). Studying postmortem samples of TS subjects, reported an increased number of dopamine uptake sites in the striatum suggesting either a greater number of DAT1 molecules or an increased number of dopamine nerve terminals (Singer et al., 1991). It is the site of action of methylphenidate (Volkow et al., 1995k) and dexedrine, compounds widely used in the treatment of ADHD. These stimulants inhibit the transport process resulting in an increase in synaptic dopamine. A significant increase in the level of dopamine transporter protein in the striatum of TD subjects vs. controls has been reported (Maison et al., 1995k). Studies of the DAT1 knockout mice, which are very hyperactive in confined spaces, showed a five fold increase in brain dopamine levels, down regulation of D.sub.2 receptors, uncoupling of D.sub.2 receptor function, and a 57% decrease in body size (Giros et al., 1996k). It is not known whether the less common DAT1 repeat alleles are associated with an increase or decrease in the number of DAT1 molecules.
An association between the 10 allele of the DAT1 gene was reported in cases of ADHD/ADD (Cook et al., 1995k), and behavioral variables in Tourette Disorder (TD) (Comings et al., 1996). The significant increase in subjects with autism is consistent with studies suggesting that TS and autism are genetically related and involve similar sets of genes (Burd et al., 1987; Comings and Comings, 1991b; Sverd, 1991).
Significant increased prevalence of the nine-repeat allele VNTR polymorphism in the 3' untranslated region of the DAT1 gene was seen in 93 alcoholics displaying withdrawal seizures or delirium, compared with 93 ethnically matched non-alcoholic controls (Sander et al., 1997). The 5' UTR 40 bp repeat polymorphism in the DAT1 was examined in subjects with drug abuse and found no significant difference in the frequency of any of the 3' UTR repeat alleles compared to normal controls (Persico et al., 1993). The 9/10 genotype has been found to associate with "pathological violent" adolescents; and the 9/9 genotype is associated in alcohol dependence with withdrawal seizures or delirium. An association between the 9 allele of the 40 bp repeat of the DAT1 gene with cocaine induced paranoia has been reported (Gelernter et al., 1994a).
Dopamine-.beta.-Hydroxylase D.beta.H is one of the major enzymes for dopamine metabolism and catalyzes the conversion of dopamine to norepinephrine (NE). In study animals, the inhibition of D.beta.H activity results in a decrease in norepinephrine levels which releases the inhibition of tyrosine hydroxylase resulting in the excessive production of dopamine. The later is associated with hyperactivity, aggression, self-stimulation, and stereotypic movements (Randrup and Scheel-Kruger, 1966; Shekin et al., 1983k). Studies of blood enzyme levels of D.beta.H have implicated a role of this enzyme in sensation seeking (Kuperman et al., 1988k; Comings et al., 1996), ADHD and conduct disorder (Rogeness et al., 1982k; Rogeness et al., 1989k).
Disturbances in dopamine-Beta-hydroxylase (D.beta.H) activity have previously been associated with childhood CD and alcoholism (Pliszka et al., 1991). It has been proposed that externalizing disorders such as CD were associated with a decrease in noradrenergic flnction and an increase in dopaminergic function, a pair of conditions that would be uniquely brought about by a D.beta.H deficiency (Quay 1986). Others reported an increased frequency of the diagnosis of CD in emotionally disturbed boys with low plasma DBD levels. However, an outpatient study by Bowden et al., 1988 found that low D.beta.H levels were much more likely in ADHD children who also had CD than ADHD children without CD (Rogeness et al., 1987; Pliszka et al., 1988; Bowden et al., 1988; Comings et al., 1996). In contrast, in outpatient studies at a juvenile detention center an association between CD and plasma D.beta.H was not found. Umberkomen et al. (1981) have shown a correlation between low D.beta.H levels and sensation-seeking behaviors.
Examination of CSF D.beta.H levels in patients with a variety of psychiatric disorders including major depression, bipolar affective disorder and schizophrenia found that the only significant correlation was between low CSF D.beta.H and bipolar affective disorder (Lerner et al, 1978). Linkage studies between the D.beta.H locus and schizophrenia (Aschauer and Meszaros, 1994), alcoholism, depression, manic-depression and Tourette's syndrome (Comings, et al., 1986) have been negative. However, some sib pair analyses suggest a weak linkage between the ABO blood group and D.beta.H, and some psychiatric disorders such as depression and alcoholism (Wilson et al., 1992).
Linkage studies between the D.beta.H locus and schizophrenia, alcoholism, depression, manic-depression, and Tourette syndrome have been negative (Aschauer and Meszaros, 1994; Comings et al., 1986). No association was found between the D.beta.HTaqIB1 allele and pathological SAB (Blum et al., 1997). The TaqI B1/B2 polymorphism was reported to be associated with controls screened to exclude drug, alcohol, and tobacco abuse. However, the B1 allele of dopamine-beta-hydroxylase gene also associated TD probands, and ADHD probands (Comings et al. (1996)).
Cannabinoid Receptors (CB1) While the association of cannabinoid receptors with the reward pathways may be primary, it is more likely that the effect is secondary through the modulating effect of anandaide and cannabinoid receptors on dopamine metabolism. This is consistent with the similarity between the results with CB1 receptors and the DRD2 receptors. Like the CB1 gene the association of genetic variants of the DRD2 gene with polysubstance abuse has been more reproducible (O'Hara et al., 1993; Smith et al., 1992; Noble et al., 1993; Comings et al., 1994) than the association with alcoholism per se. One interpretation of these observations is that the dopaminergic-cannabinoid reward pathways are activated more by drugs, especially cocaine and amphetamines, than by alcohol (DiChiara and Imperato, 1988).
Activation of the mesolimbic dopamine system is known to trigger a relapse to cocaine seeking behavior in animal models of drug dependence. This priming effect is enhanced by dopamine D.sub.2 agonists but inhibited by dopamine D.sub.1 agonists (Self et al 1996). In this regard, the ability of anandamide to cause a decrease in the ratio of D.sub.1 and D.sub.2 receptors in the striatum (Romero et al., 1995) may be the link that accounts for the role of CB1 variants in drug dependence.
Monamine Oxidase The Fnu4H1 polymorphism, associated with a T.fwdarw.C variant at position 1460, and the EcoRV polymorphism, associated with a T.fwdarw.G variant at position 941, of the MAO-A cDNA has been examined (Hotamisligil and Breakefield, 1994). Since both involved substitutions in the third base of a codon, they were not associated with amino acid substitutions. They examined 40 cell lines of known MAO-A activity. All lines that carried the Fnu4H1 C variant also carried the EcoRV G variant. When the sample was divided into two groups on the basis of lower vs higher MAO-A activity, the less common Fnu4H1 C or + allele (the inventors' 2 allele), present in 25% of the cell lines, was significantly (P=0.028) associated with the higher activity group. Lin et al. (1994) reported a significant increase in the more common MAOA Fnu4H1 T or 1 allele (Lin et al., 1994), associated with lower MAO levels (Lin et al., 1994) in manic depression, while Craddock et al. (1995) and Nothen et al. (1995) were unable to confirm this.
Vanyukov et al. (1993) examined the MAOA gene in 23 male and 34 female alcoholics compared to 31 male and 78 female controls, using a CA repeat polymorphism (Black et al., 1991). There was a trend in males (P=0.17) but not in females (P=0.8) for an association between higher molecular weight alleles (&gt;115 bp) in young substance abusers, and a marginal association of the &gt;115 bp alleles with age of onset (P=0.03). Tivol et al. (1996) have recently sequenced the exons of 40 control males who showed a &gt;100-fold variation in MAO A enzyme activity. There was remarkable conservation of the coding sequence. Only five polymorphisms were found. Of these, four involved the third codon position with no change in the amino acid sequence. The other was a neutral lys.fwdarw.arg substitution.
Nicotine Receptor Genes The gene for the CHRNA4 gene is located on chromosome 20q13.2-13.3 (Steinlein et al., 1994) and consists of 6 exons over 17 kb of genomic DNA (Steinlein et al., 1996). A Ser248Phe missense mutation in the transmembrane domain 2 of the CHRNA4 gene was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in one extended Australian pedigree (Steinlein et al., 1995). An insertion of three nucleotides (GCT) into the coding region for the C-terminal end of the M2 domain was found in a Norwegian pedigree with autosomal dominant nocturnal frontal lobe epilepsy (Steinlein et al., 1997b). Two other disorders of brain function, benign familial neonatal convulsions (Leppert et al., 1989; Malafosse et al., 1992) and low-voltage EEG (Steinlein et al., 1992) have also been linked to the region of the CHRNA4 locus. D20S19, a highly polymorphic locus, is in tight linkage with the genes for all three of these disorders (Steinlein et al., 1996).
A highly polymorphic dinucleotide VNTR polymorphism located in the first intron of the CHRNA4 gene was reported by Weiland and Steinlein (Weiland and Steinlein, 1996). Single base pair polymorphisms have also been reported (Steinlein, 1995; Phillips and Mulley, 1997; Guipponi et al., 1997; Steinlein et al., 1997a). Using three single base pair polymorphisms Steilein et al. (1997a) found no association between the CHRNA4 gene and panic disorder. Using the Ser248Phe missense mutation associated with ADNFLE and four silent polymorphisms, Steinlein et al., Ser248Phe missense mutation reported a modest increase in the frequency of the T allele of the CfoI 595 polymorphism in common idiopathic generalized epilepsies of childhood (0.085) versus controls (0.027).
Micro/minisatellite polymorphisms Studies of behavioral phenotypes associated with micro/minisatellite polymorphisms at different neuropsychiatric candidate genes have found a significant association between the shorter or longer alleles with various quantitative behavioral traits and mini- or microsatellites at the following genes: MAOA, MAOB, HTR1A, DAT1, DRD4, HRAS, HTT, OB, CNR1, GABRA3, GABRB3, FRAXA, and NO (Comings et al., 1996k; Comings et al., 1996l; Comings et al., 1996m; Johnson et al., 1997; Comings et al., 1998; Gade et al., 1997). Significant phenotypic behavioral effects with specific size alleles of the same polymorphisms of the DAT1 (Cook, 1995; Gelernter et al., 1994), DRD4 (Benjamin et al., 1996; Ebstein et al., 1996; Grice et al., 1996; Lahoste et al., 1996), HRAS (Herault et al., 1993; Eggers et al., 1995; Thelu et al., 1993), HTT (Ogilvie et al., 1996; Lesch et al., 1996), INS (Bennett et al., 1955; Kennedy et al., 1995; Pugliese et al., 1997; Vafiadis et al., 1997) and DBH (Wei et al., 1997) genes. These studies do not rule out the presence of an important role of single base pair changes in a subset of these length variants, (see below and Grice et al., 1996; Lichter et al., 1993; Krontiris et al., 1985).
The is evidence for an involvement of long triplet repeats in a variety of neurological disorders (Caskey et al., 1992) including fragile-X syndrome, Huntington's disease (Huntington's Disease Collaborative Research Group, 1993), myotonia dystrophica, Kennedy's disease, Friedreich's ataxia (Campuzano et al., 1996), and others (Caskey et al., 1992). At least five of these disorders involve intronic GAG repeats producing polyglutamine tracts in the amino acid sequence of the respective gene products.
Obesity Related Genes Previous studies have failed to identify any mutations of the human OB gene in several hundred obese individuals (Ezzel, 1995; Hamilton et al., 1995; Considine et al., 1996b). However, prior studies (Comings, 1996b; Comings et al., 1996c) have suggested that the mutations involved in polygenic disorders may be outside the exons and that the polymorphic dinucleotide repeats may themselves play a role in regulating the expression of the genes to which they are close to (Krontiris et al., 1993; Green and Krontiris, 1993; Trepicchio and Krontiris, 1992; Trepicchio and Krontiris, 1993; Bennett et al., 1955; Kennedy et al., 1995).
TaqI polymorhisms of the apolipoprotein gene (APOE-D) was found to associate with obese subjects and between the APO-D and fasting-insulin. This work suggests that that the APO-D polymorphism may be a genetic marker for both obesity and hyperinsulinemia (Vijayaraghavan et al., 1994).
Serotonin Genes Functional variants of this gene could account for the observed simultaneous increase or decreases of both serotonin and tryptophan in various disorders. Four different polymorphisms of the human TD02 gene have been identified. Association studies show a significant association of one or more of these polymorphisms and TS, ADHD, and drug dependence. The intron 6G-T variant was significantly associated with platelet serotonin levels (Comings et al, 1996a).
Multiple Gene Analysis Combined examination of the dopamine D4 receptor gene (DRD4), cannabinoid receptor gene (CNR1) and the GABAB3 receptor gene (GABRB3) explained 25% of the variance of the trait of IV drug use (Saucier et al, 1996; Comings et al., 1997; Johnson et al., 1997). It was observed that testing for both the OB and the DRD2 gene explained 22.8% of the variance of body mass index, demonstrating that polygenic factors influence body weight, while the association with psychiatric symptoms, determined by examination alone, accounted for a smaller percent (Comings et al., 1996b). Individual polymorphisms at three dopaminergic genes: TaqI A1 of the dopamine D2 receptor (DRD2), TaqIB1 of the dopamine B-hydroxylase (D.beta.H), and the 10/10 genotype of the 40 bp repeat of the dopamine transporter (DAT1) genes, were shown to have significant association with TS, ADHD, and CD (Comings, 1996).
The Role of Neurotransmitters and Amino Acid Precursors In addition to the genes thought to be involved in neurological disorders, neurotransmitters and pharmaceuticals have been studied for their roles in creating or alleviating certain psychological traits. In humans, it has been suggested that meso-prefrontal dopaminergic activity is involved in human cognition (Weinberger et al., 1988). In patients with Parkinson's disease and possibly in patients with schizophrenia, prefrontal activation during a cognitive task and with clinical signs of dopaminergic function (Weinberger et al., 1988k). Brain chemical turnover in animals have demonstrated changes in neurotransmitter levels following precursor amino acid loading, or systemic and direct central nervous system delivery (Blum et al., 1996a; Blum et al., 1996b). Animal studies implicate NE and dopamine in a wide range of attention-related behaviors involving search and exploratory activity, distractibility, response rate, discriminability and the switching of attention. Overall, the animal and human studies indicate a role for dopamine and NE in the early and late processing of information, respectively (Sara et al., 1994k). Several neurotransmitters, specifically, dopamine, serotonin, norepinephrine, GABA, glutamine, and opioid peptides which play vital roles in brain functioning and in mood regulation, can be dramatically influenced by the circulating levels of their precursor amino acid nutrients (Wurtman, 1981k). Data suggest that amino acid precursors and enkephalinase inhibitors provide a substantive effect on recovery from alcohol, cocaine, and food addictions (Blum et al., 1987a; Blum et al., 1987b; Blum et al., 1987c; Blum et al., 1989b; Blum et al., 1990; (Strandburg et al., 1996).
One important function of the catecholamine innervation of the cerebral cortex may be the control of attention. Of particular interest are the catecholamine projections to the cerebral cortex from the reticular formation, namely dopamine neurons in the ventral tegmentum of the midbrain and the NE neurons of the locus coeruleus in the upper pons. Both acetylcholinergic (ACH) and dopaminergic systems (DA) have been found to be crucial for the maintenance of accurate cognitive performance. A series of studies, examining those aspects of cognitive function, revealed by the radial-arm maze, found that these two neurotransmitter systems interact in a complex fashion (Levin et al., 1995). Choice accuracy deficit induced by blockade of either muscarinic- or nicotinic-ACH receptors. The choice accuracy deficit induced by blockade of muscarinic receptors with scopolamine can be reversed by the dopamine receptor blocker, haloperidol. The specific DAD1 blocker SCH23390 also has this effect, whereas the specific D.sub.2 blocker raclopride did not. This effect is seen with the D.sub.2 antagonist raclopride, but not with the D.sub.1 antagonist SCH23390. The D.sub.2 receptor was indicated in nicotinic actions on cognitive function by the finding that the selective D.sub.2 agonist LY1771555 reverses the choice accuracy deficit caused by mecamylamine. The effectiveness of these selective DA treatments in reversing cognitive deficits was due to ACH under-activation (Levin et al., 1990k).
Accumulating evidence suggests that serotonin may modulate cholinergic function in several regions of the mammalian brain and that these serotonergic/cholinergic interactions affect cognition. It is concluded that not all mnesic perturbations induced by concurrent manipulations of the serotonergic and cholinergic systems can be attributed to a serotonergic modification of the cholinergic system. The cognitive faculties of an organism arise from interactions among several neurotransmitters such as DA within brain structures such as, for instance, the hippocampus or the cortex, but also from influences on memory of other general functions that may involve cerebral substrates different from those classically related to mnesic functions (e.g., attention, arousal, sensory accuracy, etc.) (Cassel et al., 1995k).
Additionally, it has been determined that extrinsic modulation of hippocampal theta depends on the co-activation of cholinergic and GABA-ergic medial septal inputs. Cholinergic projections provide the afferent excitatory drive for hippocampal theta-on cells and septal GABA-ergic projections act to reduce the overall level of inhibition by inhibiting hippocampal GABA-ergic interneurons (hippocampal theta-off cells). Both activities must be present for the generation of hippocampal theta field and cellular activities. The balance between the cholinergic and GABA-ergic systems may determine whether hippocampal synchrony (theta) or asynchrony occurs (Smythe et al., 1992).
Other neurotransmitters like Norepinephrine (NE) may also play a role in learning and memory. Neuromodulatory properties of NE suggest that the coeruleo-cortical (LC) NE projection should play an important role in attention and memory processes. For example, the gating and tuning action of NE released in target sensory systems would promote selective attention to relevant stimuli at the critical moment of change (Sara et al., 1994). Other research suggest that one consequence of LC activation during stress or physiological challengers may be to increase or maintain arousal via release of both DA and NE (Page et al., 1994).
It has been reported that discharge of NE LC neurons in behaving rats and monkeys suggest a role for the LC system in regulating attentional state or vigilance (Aston-Jones et al., 1991k). Additional research on studies of NE in the dentate gyrus support a role for the LC in promoting both short- and long-term enhancement of responses to complex sensory inputs and are consistent with a role for the LC in memorial as well as attentional processes (Harley, 1988k). NE applied exogenously or released endogenously can initiate both a short- and a long-term potentiation (LTP) of the dentate gyrus.
Studies dealing with the effects of the neurokinin substance P (SP) and its N- and C-terminal fragments on memory, reinforcement, and brain metabolism. It was shown that Sp, when applied peripherally, promotes memory and is reinforcing. Most important, however, is the finding that these effects seemed to be encoded by different SP sequences, since the N-terminal SP1-7 enhanced memory, whereas C-hepta- and hexapeptidsequences of SP proved to be reinforcing in a dose equimolar to SP. These differential behavioral effects were paralleled by selective and site-specific changes in DA activity, as both SP and its C-, but not N-terminus, increased DA in the nucleus accumbens (Nac), but not in the neo-striatum. These results show that the reinforcing action of peripheral administered SP may be mediated by its C-terminal sequence, and that this effect could be related to DA activity in the NAC (Huston et al., 1991k).
In terms of dopaminergic activity, previous research has shown that bromocriptine, a D.sub.2 dopamine receptor agonist, can have a beneficial effect on visual-spatial working memory functions in normal human subjects (Kimberg et al, 1997). This form of memory, in which some aspect of a stimulus is maintained over a short interval of time, has also been found to be closely tied to prefrontal cortical function in both lesion and single unit recording studies with monkeys and in neuro-imaging studies in humans (Goldman et al., 1987k; Jonidas et al., 1993). A selective positive effect of bromocryptine, in reducing release rates in alcoholics as a function of dopamine D.sub.2 receptor genotype (Lawford, et al., 1995) has also been reported. In addition, it has been demonstrated a direct effect of dopamine antagonists on delay period activity of neurons in monkeys performing memory tasks (Williams et al., 1995k). Phentermine, a dopamine releaser, has been implicated in weight loss (Weintramb et al., 1992).
Moreover, pharmacological manipulation of brain dopamine concentration effects visual-spatial working memory in humans and in animals, the later effects localized to the prefrontal cortex. However, the effects of dopamine agonists on humans are still poorly understood. It has been hypothesized that bromocriptine would have an effect on cognitive functions associated with the prefrontal cortex via its effects on cortical dopamine receptors and on sub-cortical receptors in areas that project to the neocortex (Kimberg et al., 1997). They found that the effects of bromocriptine on young normal subjects depended on the subject's working memory capacity. High-capacity subjects performed more poorly on the drug, while low- capacity subjects improved. These results demonstrate an empirical link between a dopamine-mediated working memory system and higher cognitive function in humans. It has been shown that the DRD2 A1 allele is also associated with visual-spacial memory deficits as well (Berman et al., 1995k).
A double-blind study demonstrated that a D.sub.2 agonist bromocryptine or a placebo administered to alcoholics who were carriers of the A1 allele (A1/A1 and A1/A2 genotypes), or who only carried the A2 allele (A2/A2) reduced craving and anxiety among the A1 carriers who were treated with bromocryptine. The attrition rate was highest among the A1 carriers who were treated with placebo. The bromocryptine effect on the A1 carriers was much more robust as one approached the six wk period of treatment. Dopamine D.sub.2 agonist bromocryptine can improve higher-level cognitive functions.
Studies using sophisticated techniques in animals, including microdialysis measurements, have demonstrated changes in neurotransmitter output following precursor amino acid loading (Hernandez et al., 1988). In addition, behavioral changes have been demonstrated in animals following systemic and direct central nervous system delivery of precursor amino acids (Blum et al., 1972). While certain L-amino acids are neurotransmitter and neuromodulator precursors, their racemates, the D-anmino acids also have biological activity. In particular, D-phenylalanine, D-leucine, other D-amino acids as well as certain metabolites (e.g., hydrocinnamic acid) decrease the degradation of opioid peptides which are central to regulation of mood and behavior (Blum et al., 1977; Della Bella et al., 1980).
In some individuals scientists have described a phenylalanine deficiency (PHD) (Lou, 1994k). In this regard, phenylalanine and tyrosine constitute the two initial steps in the biosynthesis of dopamine, which, in its turn, is the metabolic precursor of NE. The extracellular phenylalanine concentration influences brain function in PHD by decreased dopamine synthesis. It has been shown to induce EEG slowing and has prolonged the performance time on neuropsychological tests. The tyrosine concentration in the CNS is reduced in PHD, possibly implying an insufficient substrate of tyrosine for catecholamine synthesis due to competition inhibition, for instance across the blood brain barrier. In experimental studies it has been shown that the synthesis and release of dopamine can be influenced by an increase in the availability of tyrosine. In PHD an extra dietary intake of three doses of tyrosine (160 mg/kg/24 h) induced a shortening of reaction time and decreased variability and in a double-blind, crossover study a similar dose has been reported to induce an improvement on psychological tests, while lower doses failed.
A combination of precursor amino acids having enkephalinase inhibition activity may be used for the treatment of cocaine dependence (U.S. Pat. No. 5,189,064). It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Maurer et al., 1988k). Chronic cocaine abuse alters attentional processing (Noldy et al., 1990k). It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Jonsson et al., 1996). However, paradoxically, chronic cocaine abuse alters attentional processing (Braverman and Blum, 1996). Although still controversial, attentional processing has been shown to be dependent on biogenic amine regulation (Lyoo et al., 1996).
Obesity and Neurological Functions Obesity generally is defined as being 20% or more over ideal body weight. Numerous methods of weight reduction have been attempted including hypocaloric balanced diets, "fad" diets, behavior modification, drugs (i.e. D-phenflouramine, phenteramine, etc.), surgery, total starvation, jaw wiring, and combinations of these methods. Most of these are short-term approaches to the problem and have been only transiently effective and some can even pose serious danger (Lockwood and Amatruda, 1984). Even if weight loss is demonstrated in the short-term, the weight usually is regained following discontinuation of the weight-loss regiment. Despite the fact that about 28% of the American population is obese, obesity is widely perceived as a food-addiction, a self-imposed condition with cosmetic rather than health indications (Kral et al., 1989; Weintraub and Bray, 1989).
An understanding is emerging from recent studies of some of the causes of obesity and the difficulties of treating this condition. Studies of twins among the Pima Indians have substantiated a strong genetic basis for obesity (Bouchard, 1989; Stunkard et al., 1990). Obesity is a heterogeneous and prevalent disorder which has both genetic and environmental components. The relationship between macro selection of various foods and familial substance use disorder (SUD) has been documented throughout the literature and neurochemical studies have supported the commonality of reinforcement through dopaminergic systems by alcohol, nicotine, cocaine, and carbohydrates (Nobel, 1998; DiChiara, 1988). In this regard, both obesity and SUD can be considered appetitive compulsions. Some genes such as the dopamine D2 receptor (DRD2), and dopamine transporter (DAT1) genes may be a risk factor not only for obesity (Noble et al., 1994; Comings et al., 1993; Blum et al., 1995a) but also for SUDs in general and other psychiatric disorders (Noble et al., 1994; Smith et al., 1992; Comings, 1994; Blum et al., 1995b; Comings et al., 1996; Cook et al., 1995). Additionally, the cloning and sequencing of the mouse ob gene and its human OB homologue raised hopes that defects in this gene may play a significant role in the cause of obesity in man and that Leptin, its gene product, would be useful in treatment (Zhang et al., 1994; Peileymounter et al., 1995). While genetic effects can act alone, in most cases the genetic profile only sets the stage defining the opportunity for a genetic-environmental interaction (i.e. dramatic increase in weight when coupled with increased food). For persons with such a genetic risk profile, obesity is a life-long condition requiring long term therapy as in other chronic diseases.
The specific causes of uncontrollable ingestive behavior for alcohol, drugs, and food (in particular, carbohydrates) are incompletely understood. Nevertheless, it is clear that these appetitive compulsive behaviors are a product of genetic predisposition and environmental insult factors. Numerous studies have implicated the interaction of opiates, opioid peptides, CCK-8, glycogen, DA, and insulin in glucose utilization and selective intake of carbohydrates (Morley and Levine, 1988; Moore et al., 1982; Morley et al., 1985; Riviere and Bueno, 1987). The primary neurotransmitters involved in eating behavior include the monoamines dopamine (DA), norepinephrine (Ne), epinephrine (EPI), and serotonin (5-HT); the inhibitory neurotransmitter gamma-aminobutyric (GABA); and a variety of neuropeptides such as the pancreatic polypeptides, opioid peptides, hormone-releasing factors, and various gut-brain peptides (for reviews see Cooper et al., 1988; Gosnell, 1987; Bouchard, 1994). There is extensive evidence for the role of a number of brain monoamines and neuropeptides in the control of normal eating behavior operating in concert at the mesolimbic reward system (Leibowitz and Hor, 1982). Analyses of cerebrospinal fluid in both humans and animals indicate specific disturbances in brain neurochemical function in association with abnormal eating patterns (Kaye et al., 1985; Kaye et al., 1984).
A study of overeaters demonstrated that study subjects taking a variant of PHENCAL.TM., which is a dietary supplement containing amino acid precursors, lost an average of 27 lbs in 90 days compared to 10 lbs lost in the control group (Blum, 1990). Finding that PHENCAL.TM. or other similar neuronutrients, (Blum et al., 1988c; Blum and Trachtenberg, 1988; Cold, 1996) with alcoholics, polydrug abusers, heroin abusers, and cocaine-dependent individuals facilitates recovery and further indicates a common mode of treatment for addiction to these diverse substances (Blum et al, 1996; Blum et al., 1997).
The Role of Nicotine Nicotine also releases dopamine, and nicotine has been found to improve memory performance in a variety of tests in rats, monkeys, and humans (DiChiara et al., 1988). Nicotine in a dose dependent fashion reduced incorrect responding on discrimination behavior in rats (Geller et al., 1970). This effect was similar to chlordiazepoxide but could not be mimicked by the stimulant caffeine (Geller et al., 1970). Nicotine, in the form of gum or skin patches (Sanberg et al., 1988; McConville et al., 1992; Sanberg et al., 1997) has been shown to be effective in the treatment of tics in some subjects with Tourette syndrome (TS), and cigarette smoking has been reported to enhance attention, arousal, learning and memory (Wesnes and Warburton, 1984; Warburton, 1992; Balfour and Fagerstrom, 1996) and to improve the symptoms of ADHD (Coger et al., 1996; Conners et al., 1996; Levin et al., 1996).
It has been reported a placebo-controlled double-blind study to determine the effect of using nicotine in the treatment of adults with ADHD (Levime et al., 1996; Conners et al., 1996). Of the 17 subjects, 6 were smokers and 11 were nonsmokers. All meet DSM-IV criteria for adult ADHD. The drug was delivered via a transdermal patch at a dosage of 7 mg/day for nonsmokers and 21 mg/day for smokers. Active and placebo patches were given in a counter-balanced order approximately 1 wk apart. Nicotine caused a significant overall improvement on the Clinical Global Impressions (CGI) scale. This effect was significant even when only the nonsmokers were considered, which indicated that it was not due merely to relief of withdrawal from regular smoking. Nicotine caused significantly increased vigor as measured by the Profile of Mood States (POMS) test, and an overall significant reduction in reaction time on Continuous Performance Test. There was also a significant reduction in indices of inattention. Nicotine improved accuracy of time estimation and lowered variability of time-estimation response curves. Since smoking is significantly more common in adults with ADHD than those without ADHD (Conners et al., 1996).
Interactions of nicotinic systems with dopamine systems may be important for this effect. A series of studies of nicotinic agonist and antagonist interactions with dopamine systems was conducted using rats in a win-shift working memory task in the radial-arm maze (Levin and Rose, 1995k). The working memory deficit caused by the nicotinic antagonist mecamylamine was potentiated by the D1/D2 DA antagonists haloperidol and the specific D.sub.2 antagonist raclopride. In contrast, the mecamylamine-induced deficit was reversed by co-administration of the D2/D3 agonist quinpirole. Nicotine also has significant interactions with dopamine drugs with regard to working memory performance in the radial-arm maze. The dopamine agonist pergolide did not by itself improve radial-arm choice accuracy. Nicotine was effective in reversing this deficit. When given together with nicotine, the D2/D3 agonist quinpirole improved RAM choice accuracy relative to either drug alone. Acute local infusion of mecamylamine to the midbrain dopamine nuclei effectively impairs working memory function in the radial-arm (Noble et al., 1998).
The Role of Chromium Salts (CrP and CrN) Trivalent chromium is a mineral essential for normal insulin function (Jeejeehboy et al., 1977; Schwartz et al., 1959). Some but not all previous research suggests that chromium supplementation may favorably alter risk factors for coronary artery disease (CAD) and non-insulin-dependent diabetes mellitus (NIDDM)(Abraham et al., 1992; Anderson et al., 1991; Donaldson et al., 1985; Glinsmann et al., 1966; Kaats et al., 1991; Levine et al., 1968; Page et al., 1991; Press et al., 1990; Roeback et al., 1991). Chromium is thought to cause these changes via its potentiating effect on insulin (Offenbacher et al., 1988).
Animal studies have supported the contention that CrP can lower insulin resistance and improve body composition (Liarn et al., 1993), one human study found positive changes in body composition with CrP supplements (Hasten et al., 1992), another reported positive, although not statistically significant changes in body composition (Hallmark et al., 1993), and a third failed to find any positive changes in body composition with CrP supplementation (Clancey et al., 1994). CrP supplementation has been indicated to improve body composition, particularly in the reduction of excess body fat (Page et al., 1992). However, previous work observing concurrent chromium supplementation and exercise training has been restricted to effects on body weight and composition, with conflicting results (Clancy et al., 1994; Evans et al., 1989; Evans et al., 1993; Hallmark et al., 1996; Hasten et al., 1992).
While there still is controversy regarding the effects of chromium salts (picolinate and nicotinate) on body composition and weight loss in general (Abraham et al., 1992; Anderson, 1995; Hallmark et al., 1993; Clancy et al., 1994; Bulbulian et al., 1996), some reports seem to support the positive change in body composition in humans (Kaats et al., 1996). In contrast, (Grant, et al., 1997; Bulbulian et al., 1996) reported weight gain with chromium picolinate with or without exercise in humans, while showing positive effects for the nicotinate salt in the same population (Kaats et al., 1992).
Chromium Picolinate (CrP) is the most heavily used, studied and promoted chromium compound, but in vitro work suggests that chromium nicotinate may be also viable in the area of weight loss and changes in body composition. Previous research has shown chromium picolinate supplementation decreasing fat mass and increasing fat-free mass (Kaats et al., 1991; Page et al., 1991). Pervious studies of exercise training have shown increases in fat free mass as well (Stefanick, 1993). Although studies with young men (Evans, 1989) and women (Hasten et al., 1992) suggest that combining exercise training with chromium picolinate supplementation increases the body composition changes that occur with exercise training, this finding has not been confirmed (Clancy et al., 1994; Hallmark et al., 1996). It has been reported that the nicotinate salt (CrN) may be even more important than the picolinate salt (Grant et al., 1997).
Nutritional Supplements in Treatment of Behavioral Disorders Perturbation of neurotransmitter actions may underlay a variety of psychiatric and behavioral disorders (Blum et al., 1996c; Persico and Uhl, 1997; Noble et al., 1991). Specifically, anomalous regulation of dopamine, serotonin, norepinephrine, gammaminobutyric acid (GABA), glutamine, and the opioid peptides are thought to play crucial roles in the addictive disorders, particularly those involving alcohol and cocaine abuse (Pohjalainen et al., 1996). Consequently, these observations have provided momentum to the idea that ingestion of selected nutrients could affect mood and therefore behavior in humans. While nutritional strategies have been employed in the past (Grandy et al., 1989), demonstrations of effectiveness have been decidedly limited. A substantive effect of a combination of amino acid precursors and enkephalinase inhibitors on recovery from certain RDS behaviors including alcohol, cocaine, and overeating have been indicated (Noble et al., 1993; Noble et al., 1994; Blum et al., 1994; Balldin et al., 1993; Duffy et al., 1994; American Psychiatric Association Task Force, 1991, U.S. Pat. No. 5,189,064).
Polygenic Analysis of Genes involved in Psychiatric and Other Polygenic Traits It has been hypothesized that psychiatric behaviors share genes in common and that once the dopamine-serotonin and other neurotransmitter balance is upset, the resulting brain dysfunction can result in a wide range of different behaviors (Comings, 1990a; Comings and Comings, 1991a; Winokur et al., 1970; Comings, 1994b; Comings, 1995b). Others have supported the proposal that personality traits may have distinct neurochemical and genetic substrates mediated by genetic variability in dopamine transmission as well as other neurotransmitters (Cloninger, 1983; Benjamin, et al., 1996, Epstein et al., 1996, Cloninger, 1991). The molecular genetic studies of the DRD2, D.beta.H, DAT (Comings et al., 1996a), and clinical genetic studies (Comings 1994b; Comings 1994c; Comings 1995b; Biederman et al., 1991; Comings and Comings 1987), indicate ADHD, Tourette's syndrome, conduct disorder, ODD, dyslexia, learning disorders, stuttering, drug dependence and alcoholism are etiologically related spectrum disorders, with male predominance.
In the past two decades a large proportion of the genes for these disorders have been identified, localized, cloned, and sequenced. As the number of such genes remaining to be identified has decreased there has been an increased interest in the more common polygenic disorders. It has often been suggested that the genes involved in these disorders will be far more difficult to identify. This difficulty is well illustrated by the psychiatric disorders. Despite large numbers of linkage studies of manic-depressive disorder, schizophrenia, Tourette syndrome, panic disorder, autism, and others, with the possible exception of bipolar disorder (Risch and Botstein, 1996), there have been few replicated findings. Many of the efforts to find the genes in complex disorders have simply attempted to force feed the single-gene single-disease model into service for polygenic disorders by using lod score analysis, other family based forms of linkage analyses, or the haplotype-relative risk technique (Falk and Rubinstein, 1987). Presently the most popular method used to identify the genes in complex disorders consists of whole genome screening of affected sib pairs. Non-parametric approaches to linkage (Weeks and Lange, 1988) are better suited to complex inheritance (but see Greenberg et al., 1996). However, when a given gene accounts for less than 8% of the variance, a large number of parent-child sets or sib-pairs must be examined (Carey and Williamson, 1991).
There has been an increased recognition that only association studies may have the power to identify genes with small contributions to the percent of variance of a given polygenic trait (Risch and Merikangas, 1996; Collins et al., 1997). Association studies, comparing the frequency of the mutant candidate genes in severely affected probands to totally unrelated, ethnically matched controls that are free of the disease, can identify these small effects (Weeks and Lathrop, 1995; Comings, 1996; Owen and McGuffin, 1993). The additive effect of the DRD2, D.beta.H and DAT genes (Comings et al., 1996j), the DRD1 and DRD2 genes (Comings et al., 1997a), the OB and DRD2 genes (Comings et al., 1996d), and other gene combinations genes in TS, ADHD, conduct disorders, stuttering, and related behaviors has been examined. In TS syndrome it has been found that identifying a role of three dopaminergic genes (DRD2, D.beta.H and DAT1) was best determined by an examination of a relatively large number of TS subjects, their relatives and controls, suggesting that TS and related disorders are polygenically inherited and that each gene contributes only a small percent of the variance of any behavior score (Comings et al., 1996a; Comings 1996b; Comings et al., 1996d; Comings 1996c).
Most psychiatric disorders are polygenic (Comings, 1996b) and that each gene accounts for less than 10% and usually less than 5% of the variance of a given behavioral variable. In both studies, the strength of the associations was increased by the examination of the additive effect of more than one gene. One of the major impediments to the wider use of association studies is the lack of availability of suitable polymorphisms at or near the many candidate genes that have been cloned and sequenced (Comings, 1994). However, even when this technique or classical linkage techniques are used, positive findings from one group of investigators are often not replicated in subsequent studies (Egeland et al., 1987; Kelsoe et al., 1989; Blum et al., 1990; Bolos et al., 1990). This technique can also produce false positives due to population stratification, however, this can be minimized using the haplotype relative risk procedure (Falk and Rubinstein, 1987) with large numbers of subjects (Comings, 1995). The small size of these effects, and the difficulties in replication have led to a feeling of pessimism about whether it will be possible to identify the genes involved in polygenic disorders (Moldin, 1997).